Methods of inhibiting neurodegenerative disease

ABSTRACT

The invention provides methods for treating neurodegenerative disease as well as methods for screening compounds for the treatment of neurodegenerative disease. Transgenic animals, e.g., insects, which may be used as a model for neurodegenerative disease, are also provided.

DESCRIPTION OF THE INVENTION

1. Field of the Invention

The invention relates generally to the field of neurodegenerativediseases, e.g., Alheimer's disease. In certain embodiments, theinvention relates to methods of treating neurodegenerative diseases.Other embodiments of the invention relate to methods of screening forcompounds which inhibit neurodegenerative diseases. Certain embodimentsof the invention provide a transgenic animal model for neurodegenerativediseases.

2. Background of the Invention

Alheimer's disease (AD) is the most frequently diagnosedneurodegenerative disease, and is characterized histologically by twodistinguishing hallmarks: senile plaques, which are extracellulardeposits of β-amyloid (Aβ₄₂), and intracellular neurofibrillary tanglesof aggregated hyperphosphorylated tau protein (Giannakopoulos etal.,1996, J. Neuropathol. Exp. Neurol. 55:1210). It has been shown thatthe accumulation of Aβ₄₂ precedes the pathological changes associatedwith neurodegeneration (Pike et al.,1993, J. Neurosci. 13:1676) and thatinhibition of Aβ₄₂ accumulation can effectively intervene in the ADpathogenesis (Cherny et al., 2001, Neuron 30: 665). Less clear is thecontribution of the downstream protein targets of Aβ₄₂toxicity inneurons. Understanding the Aβ₄₂-induced signaling cascade may provideclues for designing therapeutic strategies for treatment of AD.

One component of the signaling pathway associated with AD pathogenesisis cyclin dependent kinase 5 (Cdk5). Cdk5 is activated by Aβ₄₂, theprotein found in AD associated plaques (Alvarez et al., 2001, Exp. CellRes. 264:266). Deregulated Cdk5 is correlated with tauhyperphosphorylation which leads to one hallmark of AD associatedpathology (Noble et al., 2003, Neuron 38:555). Cdk5 requires associationwith its regulatory partners for kinase activation. p35 is one knownactivating partner, and is expressed primarily in postmitotic neurons(Nikolic et al., 1996, Genes Dev. 10:816; Tsai et al., 1994, Nature371:419). While under normal physiological conditions, Cdk5 associateswith p35 in healthy neurons, Cdk5 appears to be deregulated by itsassociation with p25, a calpain digestion product of p35 found in ADneurons (Patrick et al., 1999, J. Biol. Chem. 273:24057). It has beenhypothesized that the complex of Cdk5/p25 hyperphosphorylates tau toreduce its association with microtubules, which subsequently results inneuronal apoptosis (Zhang et al., 2002, J. Neurochem. 81:307). Neuronalapoptosis is yet another hallmark associated with AD pathology (Glabe,2001, J. Mol. Neurosci. 17:137; Yankner, 1996, Neuron 16:921).

Abl, another cellular kinase, is a nonreceptor tyrosine kinasedistributed both in the nucleus and cytoplasm of proliferating cells.Abl kinase appears to be evolutionarily conserved from fly, e.g.,Drosophila melanogaster, to human, functioning in the developing nervoussystem (Hoffmann, 1991, Trends Genet. 7:351; Van Etten, 1999, TrendsCell Biol. 9:179). Evidence suggests that Abl kinase participates in theregulation of apoptosis (Barila et al, 2003, Mol. Cell Biol. 23:2790;Wang, 2000, Oncogene 19:5643). Abl has not, however, previously beenassociated with AD pathogenesis.

While the molecular mechanisms underlying neurodegenerative diseaseshave been widely investigated, therapeutic targets for the treatment ofneurodegenerative diseases such as AD still must be identified.Screening systems to evaluate therapeutic agents that can treatneurodegenerative diseases are also needed.

SUMMARY OF THE INVENTION

The invention relates, in part, to the discovery that Abl kinase (Abl)actively participates in the pathogenesis of neurodegenerative diseasessuch as AD. Inhibiting Abl abrogates both activation of Cdk5 and itstranslocation in neurons expressing or containing Aβ₄₂, thus inhibitingneurodegeneration associated with AD.

Embodiments of the invention, provide methods of treating a subjecthaving a neurodegenerative disease, such as, e.g., AD, comprisingadministering to the subject, an agent which inhibits Abl kinaseactivity.

Certain embodiments of the invention provide methods of inhibitingdegeneration of a neuron comprising contacting the neuron with an agentwhich inhibits Abl kinase activity. In some embodiments the degenerationis apoptotic.

The invention also provides methods of inhibiting formation of a complexcomprising Abl, Cdk5, Cables, Abi and p35 or a fragment thereof in aneuron comprising contacting the neuron with an agent which inhibits Ablkinase activity.

The invention further provides methods of inhibiting Cdk5 activity in acell comprising contacting the cell with an agent which inhibits Ablkinase activity thereby inhibiting Cdk5 activity.

Certain embodiments of the invention provide methods of screening for anagent which treats a neurodegenerative disease comprising contacting acell with the agent and measuring Abl kinase activity in the cell wherea decrease in Abl kinase activity in the cell contacted with the agentcompared to a cell not contacted with the agent indicates that the agentcan be used to treat a neurodegenerative disease. In certain embodimentsthe neurodegenerative disease is AD.

In some embodiments the invention provides a method of screening for anagent which inhibits degeneration of a neuron comprising contacting acell with the agent and measuring the Abl kinase activity in the cell,where a decrease in Abl kinase activity in the cell contacted with theagent compared to a cell not contacted with the agent indicates that theagent can be used to treat neurodegeneration.

In some embodiments, the invention provides a transgenic animal,including a transgenic insect such as Drosophila melanogaster,comprising a transgene comprising a nucleotide sequence encoding a βamyloid protein, e.g., Aβ₄₂.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the suppression of Abl kinase activity and that Ablexpression prevents Aβ₄₂-induced neuronal death in Drosophila. FIG. 1Ais an immunoprecipitation and Western blot showing kinase activity inBG2-c2 cells treated with Aβ₄₂ and/or STI571 (Abl kinase inhibitor).BG2-c2 cells were treated with Aβ₄₂ (2 μM) and/or an Abl kinaseinhibitor (STI571, 20 μM) for one hour to investigate the effect onoverexpressed-Ena phosphorylation by Abl. FIG. 1B shows thatAβ₄₂-induced neuronal death was suppressed by STI571 in Drosophilaneuronal cells. Neuronal viability was determined by MTT assays. FIG. 1Cshows that Abl protein affected Aβ₄₂-induced neuronal death. dsRNA-ablwas introduced by transfection two days before treating with Aβ₄₂ for 24hours. Viability values of control group were adjusted to 100% (n=4);**: p<0.01 versus control group; ++: p<0.01 versus the Aβ₄₂ group. FIG.1D shows controls of dsRNA-abl. The efficiency and specificity ofdsRNA-abl (0˜20 μg/2×10⁶ cells) were evaluated by monitoring theexpression of HA tagged-Abl, Cdk5, and actin (upper panel). dsRNA-EGFPwas used as non-specific control (lower panel).

FIG. 2A shows a co-immunoprecipitation and Western blot of Cdk5 and Abl.Abl-Cdk5 interaction was enhanced by activation of Abl activity.Neuronal cells were transfected with Abl-HA 2 days before treatment withAβ₄₂ (2 μM) or STI571 (20 μM) for one hour. Cell lysates were subjectedto anti-HA immunoprecipitation followed by both anti-Cdk5 and anti-HAimmunoblotting. FIG. 2B shows an immunoprecipitation and Western blot ofCdk5 and Abl (top panel) and the result of an in vitro kinase assayusing the immunoprecipitates and histone H1 as a substrate (bottompanel). Elevation of Cdk5 kinase activity required Abl kinase activity.The numbers below the bottom panel indicate the relative intensities ofhistone H1 phosphorylation.

FIG. 3 shows the effects of ectopic expression of Aβ₄₂ in Drosophilaeye. Ectopic expression of Aβ₄₂ induces neurodegeneration in eyeimaginal disc and adult rough eye phenotypes, which are suppressed inAbl mutant flies. FIG. 3A shows Aβ₄₂ was ectopically expressed by theeye-specific gmr-GAL4 driver in wild-type and Abl mutant flies.Apoptotic cells in eye discs of 3rd-instar larvae were detected by TUNELassay as shown in FIG. 3A, panel b. FIG. 3B shows adult eye phenotypesexamined by scanning electron microscope (SEM). Panels e, f, g, and hare enlarged from relative positions in panels a, b, c, and d,respectively. Note the defects of bristles and facets in Aβ₄₂-expressioncompound eyes. Scale bar: 0.1 mm.

FIG. 4A shows an immunoprecipitate and Western blot of Cdk5 andphophorylated Cdk5 (Cdk5-pY15)(top panel) and the correlation withkinase activity in an in vitro assay using H1 histone as a substrateover time (bottom panel). Drosophila neuronal cells were treated withAβ42 (2 μM) for 15-60 minutes to investigate its effect on Cdk5-pY15 andCdk5 kinase activity over the time-course. FIG. 4B shows animmunoprecipitation and a Western blot of Cdk5 and phophorylated Cdk5(Cdk5-pY15)(top panel) and the correlation with kinase activity in an invitro assay using H1 histone as a substrate (bottom panel) with andwithout the Abl kinase inhibitor STI571. Inhibition of Abl kinasereduced Aβ₄₂-induced Cdk5-pY15 and Cdk5 kinase activity. STI571 (20 μM)and/or A(342 (2 μM) were added simultaneously to neuronal cells for 60minutes. FIG. 4C is similar to FIG. 4B except that STI571 has beenreplaced with double stranded RNA-abl (dsRNA-abl) or double strandedRNA-EGFP (dsRNA-EGFP) as a non specific control. Suppression of Ablprotein expression with ds-RNA-abl reduced Aβ4₂-induced Cdk5 kinaseactivity. Cdk5-pY15 was markedly suppressed (though not entirely) bytransfection of dsRNA-abl into BG2-c2 cells (20 μg/2×10⁶ cells).Comparable levels of immunoprecipitated Cdk5 were detected withanti-Cdk5 antibody. FIG. 4D shows an immunoprecipitation and a Westernblot of Cdk5 and phosphorylated Cdk5 (Cdk5-pY15)(top panel) and thecorrelation with kinase activity in an in vitro assay using H1 histoneas a substrate (bottom panel) from lysates derived from the heads oftransgenic flies expressing wild type Abl or abl mutants. Cdk5 proteinfrom different strains of fly heads were examined for Y15phosphorylation and kinase activity. The numbers below the figuresrepresent the relative Cdk5 kinase activity. GMR is a control fly notexpressing Aβ₄₂ (lane 1); GMR>Aβ₄₂ overexpresses Aβ₄₂ in a wild type Ablbackground (lane 2); Abl mutant is not overexpressing Aβ₄₂ (lane 3); andGMR>Aβ₄₂Abl mutant overexpresses Aβ₄₂ in the presence of the Abl mutant(lane 4).

FIG. 5A schematically shows the structural conservation of p35 betweenhuman and Drosophila. The putative calpain digesting site is shown inhuman p35 sequence, but the consensus sequence is not conserved inDrosophila (highlighted characters show the conserved amino acids). MS,represents the myristoylation site. CRD represents the Cdk5 regulatorydomain. FIG. 5(B) shows that Drosophila p35 is not cleaved to p25 in thepresence of Aβ₄₂ either in vitro (left panel) or in vivo (right panel).Left panel, two days after transfection of HA-tagged Dp35, Drosophilaneuronal cells were treated with 5 or 10 μM Aβ₄₂ or calcium ionophore(A23187 with 5 μM CaCl2) for an additional 24 hours. Cell lysates wereimmunoblotted with anti-HA and anti-actin antibodies to examine the Dp35cleavage and protein loading respectively. Right panel, adult fly headsectopically expressing Aβ₄₂ and/or Dp35-HA (driven by gmr-GAL4) werelysed and immunoblotted with anti-HA and anti-actin antibodies.Consistently, these results revealed no detectable cleavage or reductionof Dp35-HA protein (˜53 kDa) in either the cell culture or transgenicsystems.

FIG. 6A (left panel) is a Western blot showing that expression ofexogenous Dp35-HA is suppressed in a dose dependant manner by doublestranded RNA-Dp35 (dsRNA-DP35). The right panel shows that dsRNA-Dp35suppresses kinase activity of Cdk5 in an in vitro kinase assay usinghistone H1 as a substrate, but does not effect Cdk5 phosphorylation(Cdk5-pY15) in response to Aβ₄₂. Y15 phosphorylation level and kinaseactivity of Cdk5 were determined while Dp35 was targeted by dsRNA incells treated with Aβ₄₂. dsRNA-Dp35 was transfected into neuronal cellstwo days before Aβ₄₂ treatment. After one hour of Aβ₄₂ treatment, cellswere lysed and immunoprecipitated with anti-Cdk5 antibody to examineCdk5-pY15 and kinase activity. The Cdk5-pY15 signal was largely retainedbut its kinase activity was dramatically abolished. FIG. 6B shows theresults of a TUNEL assay of apoptotic cells in eye discs of 3rd-instarlarvae. Apoptosis in Drosophila eye discs caused by the Dp35-transgenewas mitigated in the Abl mutant background. Scale bar: 0.1mm. FIG. 6Care SEM images of adult transgenic fly compound eye phenotypescorresponding respectively to the results in 6B.

FIG. 7 shows that Aβ₄₂ affects Cdk5 intraneuronal translocalization by ap25-independent, Abl-associated phosphorylation of Cdk5-Y15 mechanism inDrosophila neuronal cells. FIG. 7A shows confocal microscopy analysis ofimmunostaining of endogenous Cdk5 (TRITC-labeled) in BG2-c2 neuronalcells. Cdk5 translocalized from the cell membranes (a) to perinuclearregions (b) following stimulation of cells with 2 μM Aβ₄₂ for one hour.Nuclei were labeled with DAPI (blue) (b). Cdk5 translocalization wasblocked by simultaneously adding 5 μM BL-1 (a Cdk5 kinase inhibitor)with Aβ₄₂ to suppressed Cdk5 kinase activation by Aβ₄₂ (c).Aβ₄₂-stimulated Cdk5 translocalization to perinuclear cytoplasm revertedback to the plasma membrane by inhibiting Cdk5 kinase activity one hourafter Aβ₄₂ treatment (d). FIG. 7B shows that phosphorylation of Cdk5-Y15is essential for Aβ₄₂-induced Cdk5 translocalization. BG2-c2 cells werecotransfected with an EGFP plasmid (panels g and h) and with Cdk5-Y15Fmutant to differentiate the mutant Cdk5-expressing cells (panel e andarrow in panel f) from the non-transfected cells. Stimulation of cellswith Aβ₄₂ for one hour markedly translocalized endogenous wild-type Cdk5protein to the perinuclear regions (arrowhead in panel f) in contrast tothat of exogenous Cdk5-Y15F protein (arrow in panel f). FIG. 7C showsthat Abl kinase activity is important for Aβ₄₂-stimulated Cdk5translocalization. Cdk5 and Abl-HA were double immunolabeled toinvestigate whether they colocalized upon Aβ₄₂ stimulus. Withouttreatment of Aβ₄₂, Abl-HA (FITC-labeled) and Cdk5 (TRITC-labeled) werecolocalized to the cell membrane and cytoplasm (panels i, l, and o). Asexpected, Abl-HA and Cdk5 colocalized to perinuclear regions in responseto Aβ₄₂ stimulus (panels j, m, and p). White arrow indicates an Abl-HAtransfected cell exhibited no Abl or Cdk5 protein translocalization toperinuclear region when cells were simultaneously treated with STI571and Aβ₄₂ (panels k, n, and q). Scale bar: 10 μm.

FIG. 8 shows that Abl functions cooperatively with p25 and active Cdk5in Aβ₄₂-induced human neuronal death. FIG. 8A shows that STI571effectively suppressed Aβ₄₂-induced IMR-32 neuronal death after 72hours-treatment with Aβ₄₂. Cells were pretreated for 24 hours withSTI571 before stimulation with Aβ₄₂ for 24 hours or 72 hours (n=4,24-hour control value is considered 100%; **: p<0.01 versus controlgroup in the same time point; ++: p<0.01 versus Aβ₄₂ group). Inductionof p35 cleavage into p25 by Aβ₄₂ is incapable of initiating Cdk5 kinaseactivity in cells lacking Abl kinase activity. Human IMR-32 cells werepretreated for 24 hours with STI571 before stimulation with/without Aβ₄₂(20 μM) for 24 hours to examine the effect on Cdk5-pY15 and Cdk5 kinaseactivity. FIG. 8B shows that the cleavage of p35 into p25 by theinduction of Aβ₄₂ was not noticeably affected by STI571, but Cdk5 kinaseactivity and Y15 phosphorylation was suppressed by STI571 treatment.FIG. 8C shows that co-treatment of calpain inhibitor (Calpeptin) andSTI571 further reduced Cdk5 activity compared to individual treatment(lane 5 vs. lane 3 and 4). The numbers below the figures represent therelative Cdk5 kinase activity. FIG. 8D is a schematic diagram of theAβ₄₂-triggered neurodegeneration model showing the role for Abl inderegulation of Cdk5 kinase activity and subcellular localization.

DESCRIPTION OF THE EMBODIMENTS

The invention is based, in part, on the discovery that Abl kinase (Abl)actively participates in the pathogenesis of neurodegenerative diseases,such as, e.g., AD. As a result of this discovery, methods of treatingneurodegenerative diseases can be developed based on inhibition of Ablwhich reduces neurodegeneration associated with diseases such as AD.

A. Definitions

“Amyloid precursor protein” (APP), as used herein, refers to aheterogeneous group of ubiquitously expressed polypeptides migratingbetween 110 and 140 kDa on electrophoretic gels. This heterogeneityarises both from alternative splicing (yielding 3 major isoforms of 695,751, and 770 residues) as well as by a variety of posttranslationalmodifications, including the addition of N- and O-linked sugars,sulfation, and phosphorylation. The APP splice forms containing 751 or770 amino acids are widely expressed in nonneuronal cells throughout thebody and also occur in neurons. Neurons express higher levels of the695-residue isoform. APP is the precursor of Aβ₄₂, associated with ADplaques. The cleavage of amyloid precursor protein by three differentprotease activities termed α-, β-, and γ-secretase is the decisive eventby which either the pathogenic amyloid β (Aβ₄₂) peptides associated withAD or the harmless (and possibly even beneficial) APPsα fragments arereleased. In humans the gene encoding APP is found on chromosome 21.

“Aβ₄₂,” as used herein, refers to a cleavage product of APP comprising(SEQ ID NO: 3). In some cases Gly may be substituted for Ala at position21 of SEQ ID NO: 3. In some cases Gin may be substituted for Glu atposition 22 of SEQ ID NO: 3. In some cases Val may be substituted forAla at position 42 of SEQ ID NO: 3.

“Abl,” as used herein, refers to a non-membrane bound tyrosine kinasefound in a variety of cell types including neurons. In one activatedform, Bcr-Abl, it is oncogenic causing various forms of leukemia inhumans. Phosphorylation of tyrosine is one activity associated with Abland the specific inhibition of this activity with STI571 is used totreat leukemia. Abl is described in greater detail infra.

“Alheimer's disease,” (AD) as used herein, refers to a progressivedegenerative disease of the brain that causes impairment of memory anddementia manifested by confusion, visual-spatial disorientation,inability to calculate and deterioration of judgment. Atrophy of thecerebral cortex, with consequent enlargement of sulci and ventricles maybe grossly evident on imaging studies. Histologically the cortex,hippocampus and amygdala show atrophy of neurons, with cytoplasmicvacuoles and argentophillic granules; distortion of intracellularneurofibrils (neurofibrillary tangles) due to excessive phosphorylationof microtubular tau proteins; and plaques composed of granular orfilamentous argentophillic masses with a core of the 42 amino acid formof β amyloid (Aβ₄₂). The concentration of tau protein in thecerebrospinal fluid is increased, while the concentration of Aβ₄₂ isdecreased.

“Cdk5,” as used herein, refers to cyclin dependent protein kinase 5.Cdk5 is a serine/threonine kinase which requires p35 (or p25) bindingfor activation. Cdk5 plays a role in neuritic outgrowth, corticallamination and synaptogenesis.

“Degeneration of a neuron,” as used herein, refers to the cell death ofa neuron, either in vivo or in vitro. The cell death may, in some cases,be the result of apoptosis.

“Erbstatin analog,” as used herein, refers to non-specific inhibitors oftyrosine kinase activity in epidermal growth factor (EGF) and Abl. Oneexample is 2,5-dihydroxymethylcinnamate which is commercially available.

“Kinase,” as used herein, refers to an enzyme which can catalyze thetransfer of phosphate groups usually to a tyrosine, threonine or serineresidue. Protein phosphorylation is a basic mechanism for modificationof protein function in eukaryotic cells. Phosphorylation anddephophorylation of proteins is a chemical signaling mechanism used bycells to relay messages from the outside environment (i.e., outside thecell membrane) to the interior of the cell. Typically the signal isultimately transferred to the nucleus where it alters gene expression.Tyrosine kinases are involved in immune, endocrine and nervous systemphysiology and pathology.

“Nucleic acid,” as used herein, refers to polymers comprised ofdeoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form. The term “nucleic acid” encompasses nucleic acidscontaining naturally occurring nucleotides as well as analogues ofnatural nucleotides that have binding properties similar to thereference nucleic acid. The term nucleic acid also includes cDNA or anmRNA encoded by a gene. A nucleic acid will be able to hybridize to itscomplement through complementary base pairing, e.g., via a hydrogenbond.

An “oligonucleotide,” as used herein, refers to a single-strandednucleic acid that typically is less than or equal to 100 bases long. Ofcourse, complementary oligonucleotides may be annealed to formdouble-stranded nucleic acids. As used herein, an oligonucleotide mayinclude natural (i.e., A, G, C, T, or U) or modified bases. In addition,the bases in an oligonucleotide may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere with interstrandbase pairing. Thus, for example, oligonucleotides may be peptide-nucleicacids in which the constituent bases are joined by peptide bonds ratherthan phosphodiester linkages (see, e.g., Nielson, 2001, Current Opinionin Biotechnology 12:16). Optionally, the oligonucleotides may bedirectly labeled with a detectable substance such as radioisotopes,chromophores, lumiphores, chromogens, or ECL moieties or may beindirectly labeled, for example, with biotin to which a streptavidin oravidin complex may later bind.

“Neurodegenerative disease,” as used herein, refers to any conditioncharacterized by the progressive loss of neurons, due to cell death, inthe central nervous system of a subject.

“Polypeptide”, as used herein, refers to a polymer of amino acids anddoes not refer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term does not exclude post-expression modifications ofthe polypeptide, for example, glycosylation, acetylation,phosphorylation, pegylation, addition of a lipid moiety, or the additionof any organic or inorganic molecule. Included within the definition,are for example, polypeptides containing one or more analogs of an aminoacid (including, for example, unnatural amino acids) and polypeptideswith substituted linkages, as well as other modifications known in theart, both naturally occurring and non-naturally occurring.

“Specific binding partner,” as used herein, refers to a first moleculethat can form a relatively stable complex with a second molecule underphysiologic conditions. In general, specific binding is characterized bya relatively high affinity and a relatively low to moderate capacity.Nonspecific binding usually has a low affinity with a moderate to highcapacity. Typically, binding is considered specific when the affinityconstant K_(a) is higher than about 10⁶M⁻¹, or is higher than about10⁸M⁻¹. A higher affinity constant indicates greater affinity, and thusgreater specificity. Antibodies typically bind antigens with an affinityconstant in the range of 10⁶M⁻¹ to 10⁹M⁻¹ or higher. If desired,nonspecific binding may be reduced without substantially affectingspecific binding by varying the binding conditions. Such conditions areknown in the art, and a skilled artisan using routine techniques canselect appropriate conditions. The conditions may be defined, forexample, in terms of molecular concentration, ionic strength of thesolution, temperature, time allowed for binding, concentration ofunrelated molecules (e.g., serum albumin, milk casein), etc.

“Subject”, as used herein, means an animal or an insect including ahuman or non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, asheep, a pig, a goat, a non-human primate, or an insect, such as a fly.

“Transgene,” as used herein, refers a gene engineered by the human handto be expressed in an organism such as unicellular organism, e.g., aprokaryotic or eukaryotic cell, or a multicellular organism such as aplant, a fungus or an animal, e.g., a mammal, an insect.

“Treat,” “treatment,” and “treating,” as used herein refer to any of thefollowing: the reduction in severity of a disease or condition; thereduction in the duration of a disease course; the amelioration of oneor more symptoms associated with a disease or condition; the provisionof beneficial effects to a subject with a disease or condition, withoutnecessarily curing the disease or condition; the prophylaxis of one ormore symptoms associated with a disease or condition.

B. Pathological Events in Alheimer's Disease

Two events are associated with the AD pathology. The first is theaccumulation of β amyloid, e.g., Aβ₄₂, plaques in the brain. The secondis the hyper-phosphorylation of the protein tau.

Aβ₄₂ is a cleavage product of APP. APP is first cleaved by α or βsecretase generating a C terminal fragment (C₉₉ or C₈₃) which is thencleaved by γ secretase (presenilin complex) to generate β amyloid (Aβ₄₂or Aβ₄₀). Aβ₄₂ is more hydrophobic and thus more apt to aggregate intoplaques. The Aβ₄₂ isoform is considered to be the pathogenic agent inAD.

Tau hyperphosphorylation has been implicated in altering the associationof tau and cellular microtubule proteins leading to cytoskeletalabnormalities (Lee et al., 2000, Nature 405:360). It is believed thisdisruption leads to apoptotic neuronal cell death. Aβ₄₂ activates theserine/threonine protein kinase Cdk5. Cdk5 deregulation has beenimplicated in tau hyperphophorylation. Cdk5 association with p25, acalpain cleavage product of cellular protein p35, is believed tocontribute to the aberrant activation and translocation of Cdk5 from thecell membrane to the perinuclear cytoplasm. These events are associatedwith tau hyperphosphorylation and AD pathogenesis.

The invention is based in part on the discovery that the uncleaved p35protein can associate with and activate Cdk5. Interestingly, p35 hasbeen demonstrated to be resistant to calpain-mediated cleavage afterbeing phosphorylated by Cdk5 in the developing rat brain (Saito et al.,2003, J. Neurosci. 23:1189). It has been suggested that Cdk5 inaccompany with p35 could be transported into nucleus in primary corticalneurons to facilitate apoptosis (Gong et al., 2003, Neuron 38:33;Weishaupt et al., 2003, Cell Tissue Res. 312:1). Cdk5-p25 is a morestable complex with a longer half-life that prolongs the activation ofCdk5 (Patrick et al., 1999, Nature 402:615). The invention is furtherbased in part on the discovery that the formation of p25 does notsufficiently trigger Cdk5 activity when Abl activation is blocked,indicating a critical role for Abl in the neurodegenerative process.

C. Abl Kinase

Abl is a nonreceptor protein tyrosine kinase found in the inner leafletof the plasma membrane, cytosol, endosomal membranes and nucleus. Ablkinase activity and subcellular localization are tightly regulated innormal physiology. Deregulation of Abl kinase has been implicated inseveral diseases, for example, the Bcr-Abl fusion oncoprotein is thedisease hallmark of the chronic myelogenous leukemia (CML). The Bcr-Ablfusion protein results from a chromosomal translocation and plays acausative role in certain human leukemias. Abl kinase has also beenimplicated in the regulation of apoptosis (Wang, 2000, Oncogene 19:5463;Barila et al., 2002, Mol. Cell. Biol. 23:2790). Abl has been reported tomodulate F-actin cytoskeleton and neurite extension (Woodring et al.,2002, J. Cell Biol. 156:879). Abl is activated in response to oxidativestress (Sun et al., 2000, J. Biol. Chem. 275:17237). The nuclear form ofAbl is positively regulated by genotoxic stress, such as DNA damage(Kharbanda et al., 1995, J. Biol. Chem. 270:30278).

One substrate of Abl is the cellular protein Ena. Together, they play arole in cytoskeletal regulation during cell-cell adhesion. Ena regulatesactin dynamics and cell motility of fibroblasts.

Reports suggest that Abl activates Cdk5 during brain development byphosphorylating tyrosine-15 (Y15) on Cdk5. Abl activation of Cdk5 ismediated by the adaptor protein Cables (Zukerberg et al., 2000, Neuron26:633). Another important adaptor protein is Abi. Cables can form atri-complex with Abl and Cdk5. Complex formation can result ininitiation of Cdk5 kinase activity. Abi is considered as a kinasesubstrate and a positive kinase regulator of Abl. Thus adaptor proteinsof Abl may be implicated in the regulation of Cdk5 kinase activity in ADpathogenesis.

It has now been discovered that Abl may form a complex comprising Cdk5and p35 or a fragment thereof, e.g., p25, in neurons expressing orcontaining Aβ₄₂. Abl may also contribute to the aberrant activation ofCdk5 and may cause Cdk5 to translocate from the membrane to theperinuclear region in neurons expressing or containing Aβ₄₂. Thetriggering of each of these events is associated with the pathogenesisof AD.

This invention demonstrates for the first time that Abl is essential forAβ₄₂-triggered neurodegeneration both in vivo and in vitro and that Ablcan serve as a molecular therapeutic target to stop the progress of ADpathogenesis. The anti-leukemic agent Abl kinase inhibitor, STI571,originally designed to suppress oncogenic Abl kinase activity in chronicmyelogenous leukemia is shown herein to prevent the Aβ₄₂-inducedneurodegeneration in both Drosophila and mammalian cells.

D. Abl Inhibitors

An Abl inhibitor may be any agent which inhibits at least one activityassociated with Abl involved in a cellular signaling pathway leading toneurodegeneration in vivo, i.e., in a subject, or degeneration of aneuron in vitro. An activity associated with Abl may include thephosphorylation of a substrate. The substrate may be a polypeptide suchas another kinase or alternatively the substrate may be Abl itself.

Upon activation, some kinases translocate within the cell. As anexample, Cdk5 translocates from the cell membrane to the perinuclearcytoplasm, in the presence of Aβ₄₂. The translocation is believed to beassociated with neurodegeneration seen in AD. Moreover, inhibiting Ablcan inhibit the downstream translocation of Cdk5. Thus, in someembodiments the Abl inhibitor may inhibit cellular translocation of akinase capable of participating in neurodegeneration, such as Cdk5.

Kinases may bind to specific binding partners within a cell to formcomplexes comprised of intracellular signaling molecules. As an example,in the presence of Aβ₄₂, a complex comprising Abl, p35, Cdk5 may formand lead to neurodegeneration. Adaptor proteins, e.g., Abi and Cableswhich bind Abl can be a part of the complex. In certain cell types,e.g., human neurons, calpain may cleave p35 to form p25 thereby allowingAbl, p25, and cdk5 to form a more stable complex resulting inneurodegenration. In certain embodiments the invention provides Ablinhibitors which prevent complex formation and thereby inactivate akinase, e.g., Abl, or Cdk5. Inhibitors can include specific bindingpartners of any of the proteins which form the complex, e.g., Abl, p35,Cdk5, Abi, and Cables.

Any type of molecule may be used as an Abl inhibitor. The molecule maybe a polypeptide, e.g., a specific binding partner. The molecule may bea small organic or inorganic molecule, e.g., imatinib mesylate, i.e.,STI571,6-(2,6-dichlorophenyl)-8-methyl-2-(3-methylsulfanylphenyl-amino)-8H-pyridol[2,3-d]pyrimidin-7-one,or an erbstatin analog. In certain embodiments the small molecule, e.g.,STI571, may be modified, made more lipophollic, formulated as ananoparticle, to facilitate penetration of the blood brain barrier. Themolecule may be a nucleic acid, e.g., DNA, RNA or an oligonucleotidecomprised of DNA or RNA. The nucleic acid may be single stranded ordouble stranded. The nucleic acid may hybridize to cellular DNA or RNAencoding a kinase, e.g., Abl and thereby inhibit transcription ortranslation of the gene encoding the kinase.

In some embodiments an Abl inhibitor may be an analog of a specificbinding partner of Abl. An analog of a specific binding partner mayinclude specific binding partners that have been chemically modified sothat they retain their binding activity but prevent at least one ofphosphorylation of a substrate, translocation of a kinase such as Cdk5,or formation of a complex comprised of Abl. In other embodiments aspecific binding partner may be used as an Abl inhibitor, e.g., acompound which specifically binds to Abl and inhibits at least oneactivity associated with Abl.

In some embodiments the agent may be a double stranded RNA molecule. Inspecific embodiments the double stranded RNA molecule may encode Abl.The RNA molecule can encode a full length Abi protein or a fragment ofan Abl protein so long as it inhibits the cellular expression of Ablwhen it is administered in vivo to a subject or in vitro to a cell.

In one specific embodiment, the Abl inhibitor is a nucleic acid that canbe used in RNA interference (RNAi ), e.g., short hairpin RNA (shRNA).RNAi is an evolutionarily conserved phenomenon in which gene expressionis suppressed by the introduction of homologous double-stranded RNAs(dsRNAs). After dsRNAs are delivered to the cytoplasm of the cell, theyare cleaved by the enzyme Dicer to 21-23 nucleotide small interferingRNAs. These siRNAs are then incorporated into a protein complex, theRNA-induced silence complex (RISC). The antisense strand of the duplexsiRNA guides the RISC to the homologous mRNA where the RISC associatedendoribonuclease cleaves the target RNA (see e.g., Zheng et al., 2004,Proc. Natl. Acad. Sci. USA 101:135; U.S. patent Publication Nos.20040002077, 20040018176, 20030092180, 20040023390, and 20030068821).

In another embodiment the nucleic acid Abl inhibitor can be ananti-sense molecule or a ribozyme. Antisense RNA and DNA molecules actto directly block the translation of mRNA by hybridizing to targetedmRNA and preventing protein translation. Antisense approaches involvethe design of oligonucleotides that are complementary to a target genemRNA. The antisense oligonucleotides will bind to the complementarytarget gene mRNA transcripts and prevent translation. Absolutecomplementarily, is not required.

A sequence “complementary” to a portion of an RNA, as discussed herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Antisense nucleic acids should be at least six nucleotides in length,and are preferably oligonucleotides ranging from 6 to about 50nucleotides in length. In specific aspects, the oligonucleotide is atleast 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides,or at least 50 nucleotides.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as polypeptides (e.g., for targeting hostcell receptors in vivo), or agents facilitating transport across thecell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.USA 86:6553; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648;WO 88/09810,) or the blood-brain barrier (see, e.g., WO 89/10134),hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988,BioTechniques 6:958) or intercalating agents (see, e.g., Zon, 1988,Pharm. Res. 5:539). To this end, the oligonucleotide may be conjugatedto another molecule, e.g., a polypeptide, hybridization triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent.

Ribozyme molecules designed to catalytically cleave target gene mRNAtranscripts can also be used to prevent translation of target gene mRNAand, therefore, expression of target gene product (see, e.g., WO90/11364; Sarver et al., 1990, Science 247:1222).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA (see Rossi, 1994, Current Biology 4:469). The mechanismof ribozyme action involves sequence specific hybridization of theribozyme molecule to complementary target RNA, followed by anendonucleolytic cleavage event. The composition of ribozyme moleculesmust include one or more sequences complementary to the target genemRNA, and must include the well known catalytic sequence responsible formRNA cleavage. The catalytic sequence is described, e.g., in U.S. Pat.No. 5,093,246.

In one embodiment, ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy target gene mRNAs. Inanother embodiment, the use of hammerhead ribozymes is contemplated.Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA have the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully in Myers,1995, Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, New York, and in Haseloff and Gerlach, 1988,Nature, 334:585.

E. Transgenic Animals

A growing body of evidence has suggested that AD-related molecularfactors are highly conserved in Drosophila, including the presence ofamyloid precursor protein-like (Appl), γ-secretase, presenilin, Cdk5,and tau (Guo et al., 2003, Hum. Mol. Genet. 12:2669; Hellmich et al.,1994, FEBS Lett. 356:317; Muqit and Feany, 2002, Nat. Rev. Neurosci.3:237). Thus, Drosophila may provide a powerful system for elucidatingcellular mechanisms of human neurodegenerative diseases, such as AD(Chan and Bonini, 2000, Cell Death Differ. 7:1075).

The invention now provides for transgenic animals which undergoneurodegeneration that is similar to the neurodegeneration seen in AD. Atransgenic animal is one engineered by humans to express an exogenousgene, i.e., one that is not expressed by the animal without humanintervention. The animal may be Drosophila melanogaster and thetransgene may be a gene encoding Aβ₄₂ The Aβ₄₂ may be human Aβ₄₂. Insome embodiments, the transgene is ectopically expressed. In someembodiments the transgene is expressed in the compound eye. Methods ofmaking transgenic animals which allow for the targeted controlledexpression of the desired transgene have been described (see, e.g.,Brand and Perrimon, 1993, Development 118:401). Thus in some embodimentsthe transgenic animal is comprised of the yeast trans-activator GAL4.The transgenic animal may be wild type in all respects other than thetransgene or alternatively it may contain one or more mutations withinits genome, e.g., within the Abl gene. In a specific embodiment thetransgenic animal is a Drosophila melanogaster which expresses humanAβ₄₂. In another specific embodiment the transgenic animal is aDrosophila melanogaster which expresses human Aβ₄₂ and also contains atleast one mutation in the Drosophila Abl gene. A mutation may include asubstitution of at least one nucleotide in the gene sequence, thedeletion of at least one nucleotide in the gene sequence, or theaddition of at least one nucleotide in the gene sequence or acombination of any of these. The transgene may also contain any of thedescribed mutations, as long as at least one activity of the proteinencoded by the transgene is maintained.

The transgenic animal may be used to study neurodegenerative diseases,e.g., AD. The transgenic animal may also be used to screen for compoundsthat inhibit neurodegeneration and could, thus, treat AD. The transgenicanimal may further be used to screen for compounds that inhibit Ablkinase activity.

Screening Assays

The invention provides for screening assays to identify Abl inhibitors.The inhibitors may be used to treat AD. The inhibitors may be used toinhibit neurodegeneration.

The assay may be a cell based assay. The cell may be of mammalianorigin, e.g., human. The cell may be of insect origin, e.g., Drosophilamelanogaster. In some embodiments the cell may be a neuron. The cell maybe from a cell line, e.g., BG2-c2, IMR-32 or the cell may be a primarycell derived directly from an organism such as an insect or mammal.

In certain embodiments screening assay will identify agents whichinhibit at least one activity associated with Abl. In one embodiment theactivity inhibited by the test agent will be a kinase activity, i.e.,phosphorylation of a substrate. The substrate may be any polypeptidewhich can be phosphorylated by Abl. The substrate may be a polypeptideendogenous to a cell used in the assay or alternatively it may be apolypeptide exogenous to a cell used in the assay, e.g., engineered bythe human hand. Phosphorylation can be determined using ananti-phospho-tyrosine antibody in a Western blot or animmunoprecipitation assay, or a combination of both.

Because Abl can inhibit the cellular translocation of Cdk5, anothercellular kinase, other embodiments of the invention will assay for theability of a test agent to inhibit translocation of Cdk5. The assay canbe used to screen for agents which inhibit translocation of Cdk5 withina cell, e.g., from the membrane to the perinuclear cytoplasm. Screeningfor the translocation of Cdk5 may be done using immunohistochemicaltechniques known in the art (see, e.g., Harlow et al., 1988, AntibodiesA Laboratory Manual, Cold Spring Harbor Laboratory Publications, ColdSpring Harbor).

In yet another embodiment the assay may be used to screen for inhibitorswhich prevent formation of a complex comprising Abl, and any of Cdk5,Cables, Abi and p35 or a fragment thereof. Detection of the inhibitionof the complex formation may be done by immuno-precipitation and Westernblot.

In other embodiments, an in vitro kinase assay may be used to screen forAbl inhibitors. In vitro kinase assays are known in the art (see, e.g.,Gaston et al., 2004, Exp. Hematol. 32:113). Abl can be recombinantlyexpressed and purified and combined in vitro with a substrate, e.g.,Crk, Cbl. A test agent may be added and the phosphorylation of thesubstrate can be compared to the phosphorylation of the substrate underidentical conditions without the test agent. A decrease inphosphorylation of the substrate would indicate that the test agentinhibits Abl.

G. Treatment Modalities

The Abl inhibitors of the invention can be administered intravenously,subcutaneously, intramuscularly, or via any mucosal surface, e.g.,orally, sublingually, buccally, sublingually, nasally, rectally,vaginally, or via pulmonary route. The Abl inhibitors can be implantedwithin or linked to a biopolymer solid support that allows for the slowrelease of the inhbitor to the desired site.

The dose of the Abl inhibitor will vary depending on the subject andupon the particular route of administration used. Dosages can range from0.1 to 100,000 μg/kg body weight. In one embodiment, the dosing range is0.1-1,000 μg/kg body weight. In another embodiment the dosing range is0.01-500 μg/kg body weight. The inhibitor can be administeredcontinuously or at specific timed intervals. In vitro assays may beemployed to determine optimal dose ranges and/or schedules foradministration. In vitro assays that measure kinase activity are knownin the art, e.g., immunoprecipitation and Western blot usingphospho-tyrosine specific antibodies. Additionally, effective doses maybe extrapolated from dose-response curves obtained from animal or insectmodels.

In certain embodiments the invention contemplates the administration ofan nucleic acid as an Abl inhibitor. Conventional gene transfer methodsmay be used to introduce DNA or RNA into target cells. The precisemethod used to introduce the nucleic acid is not critical to theinvention. For example, physical methods for the introduction of DNAinto cells include microinjection and. electroporation. Chemical methodssuch as co-precipitation with calcium phosphate and incorporation of DNAinto liposomes are also standard methods of introducing DNA intomammalian cells. DNA may be introduced using standard vectors, such asthose derived from, human, murine and avian retroviruses. Other viralvectors include adeno virus and adeno associated virus (see, e.g.,Gluzman et al., 1988, Viral Vectors, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.). Standard recombinant DNA methods are well known inthe art (Ausubel et al., 1989, Current Protocols in Molecular Biology,John Wiley & Sons, New York) and viral vectors for gene therapy havebeen developed and successfully used clinically (Rosenberg et al., 1990,N. Engl. J. Med. 323:370).

The invention also provides a pharmaceutical composition comprising anAbl inhibitor and a pharmaceutically acceptable carrier or excipient.Examples of suitable pharmaceutical carriers are described in E. W.Martin, 1990, Remington's Pharmaceutical Sciences, 17th Ed., Mack Pub.Co., Easton, Pa. Examples of excipients can include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol, and the like. Thecomposition can also contain pH buffering reagents, and wetting oremulsifying agents.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid for example a syrup or asuspension. The liquid can include suspending agents (e.g., sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil,oily esters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbicacid). The preparations can also include flavoring, coloring andsweetening agents. Alternatively, the composition can be presented as adry product for constitution with water or another suitable vehicle.

For buccal and sublingual administration the composition may take theform of tablets, lozenges or fast dissolving films according toconventional protocols.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from a pressurized pack or nebulizer (e.g., in PBS), with asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The pharmaceutical composition can be formulated for parenteraladministration (i.e. intravenous or intramuscular) by bolus injection.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multidose containers with an added preservative. Thecompositions can take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., pyrogen free water.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

EXAMPLES Example 1 Cell Culture, DNA Constructs and Transfection Methods

Drosophila neuronal cell line, BG2-c2 was used. It is a Drosophilamelanogaster 3rd-instar larval neuronal cell line and was cultured in M3medium (Sigma, St Louis, Mo.) plus 10% FBS (Hyclone, Logan, Utah), 10pg/ml insulin (Sigma, St Louis, Mo.), and penicillin/streptomycin(BRL-Gibco) in 24.5° C. IMR-32 (human neuroblastoma) was obtained fromATCC (ATCC, Manassas, Va.) and propagated in MEM medium (BRL-Gibco,Carlsbad, Calif.) plus non-essential amino acids (BRL-Gibco, Carlsbad,Calif.) and 10% FBS (Hyclone, Logan, Utah).

The Drosophila constitutive expression vector used for all DNAconstructs was pAc5.1 (Invitrogen, Carlsbad, Calif.). cDNA of Abl, andEna in pPac vector were subcloned into pAc5.1. Drosophila Cdk5 wasobtained from Drosophila EST LD01910 (GenBank Accession Number) andsubcloned into pAc5.1 vector by digesting with restriction enzymes Not1and Xba1. Dp35 was obtained from EST HL 05519 (GenBank Accession Number)and subsequently cloned into an HA-tagged pAc5.1 vector. Cdk5Y15F mutantwas constructed by PCR-based mutagenesis. Transient transfection ofneuronal cells was performed using the liposome-based method with 1˜2 μgDNA/105 cells. BG2-c2 cells were transfected with dsRNA (20 μg/2×106cells) two days before drug treatments for another day.

Example 2 Cell Viability Assay

The modified MTT assay (Sigma, St Louis, Mo.) was used to quantifyneuronal cell viability (Lee et al., 2000, Nature 405:360). Theintensities were measured using an optical density reader(SpectroMAXplus, Molecular Devices, Sunnyvale, Calif.) at 570 nm(background: 630 nm) and proportionally represented the viable cellnumbers.

Example 3 Immunoprecipitation and Western Blot Analysis

Cell lysate was produced in lysis buffer or extract buffer (Juang andHoffmann,1999, Oncogene 18:5138) for Cdk5 immunoprecipitation. Proteinswere analyzed by direct Western blotting (30 μg/lane) or blotting afterimmunoprecipitation (1˜2 mg/immunoprecipitation). Immunoprecipitateswere collected by binding to protein G PLUS/Protein A-Agarose (OncogeneResearch Products, Nottingham UK). The antibodies used are listed below:anti-Cdk5 antibody (clone DC17), (Upstate, Charlottesville, Va.);sc-750, (Santa Cruz Biotechnology, Inc., Santa Cruz Calif.),anti-Cdk5-pY15 antibody (sc-1 2919),(Santa Cruz Biotechnology, Inc.,Santa Cruz C), anti-HA antibody (BAbCO, Berkely, Calif.), anti-p35antibody (sc-820), (Santa Cruz Biotechnology, Inc., Santa Cruz Calif.),and affinity-purified Ena polyclonal antibody (generated by Dr. F. M.Hoffmann' Lab) (McArdle Laboratory for Cancer Research and Laboratory ofGenetics, University of Wisconsin-Madison) ECL detection reagent (NEN,Boston, Mass.) was used to detect the immunoreactive proteins.

Example 4 In vitro Cdk5 Kinase Assay

A kinase assay was performed by washing immunoprecipitates three timeswith kinase reaction buffer (50 mM HEPES [pH 7.0], 10 mM MgCl2, and 1 mMDTT). The beads with target proteins were incubated with kinase reactionbuffer containing 2 μg of substrate (histone H1)(Calbiochem, San Diego,Calif.) and 10 μCi of ³³Pγ-ATP in a final volume of 40 μl at 30° C. for30 minutes.

Example 5 Double Stranded RNA Synthesis

DNA fragments including both sense and antisense coding sequences forDrosophila abl (729 bp, CDS: 241˜969), EGFP (full-length, 765 bp,Invitrogen, Carlsbad, Calif.), and Dp35 (720 bp, CDS: 1-720) wereamplified by PCR. PCR products were subcloned into the vector (all cutwith BamH1 and blunt-end products were produced), pBluescript, cut byEcoRV, (Strategen, Wakefield, Wash.) and subsequently used as a templatefor in vitro transcription by T3/T7 MEGAscript kit (Ambion, Austin,Tex.). Single stranded RNA was generated and annealed with compensatorystrands in 65° C. for 30 minutes, followed by 94° C., for 5 minutes and72° C., for 10 minutes. Double stranded RNA products were confirmed as asingle band by electrophoresis and stored at −20° C.

Example 6 Immunohistochemistry

Neuronal cells cultured on coverslips (coated by poly-L-lysine)(Biochrom AG, Berlin, Germany) were fixed, permeabilized, and blocked asprevious described (Gertler et al., 1995, Genes Dev. 9:521). Primaryantibodies (anti-Cdk5, 2 μg/ml; anti-HA, 1:500) diluted in 3% BSA-PBSwere incubated with coverslips overnight at 4° C. Cells were washed inPBS and exposed to FITC or TRITC-conjugated secondary antibodies (bothaffinity-purified goat anti-mouse lgG, 1:2000, (Jackson ImmunoResearchLaboratories, Inc, West Grove, Pa.) for one hour at room temperature.After extensive washing, coverslips were mounted in Gel/Mount (Biomeda,Foster City, Calif.) and observed by Leica confocal microscopy. TheTUNEL (TdT-mediated dUTP nick end labeling) assay was carried out usingthe In Situ Cell Death Detection Kit™ (Roche, Nutley, N.J.).Third-instar larvae were dissected in M3 medium and eye imaginal discswere fixed in 4% paraformaldehyde/PBS for 20 minutes at room temperatureand then washed three times with PBS-T (PBS/0.3% Triton X-100). Thesamples were incubated in the TUNEL reaction mixture (includingFITC-conjugated modified nucleotides and terminal deoxynucleotidyltransferase) for one hour at 37° C. TUNEL signals were visualizeddirectly under Leica confocal microscopy with a FITC filter.

Example 7 Generation of Transgenic Flies

A cDNA encoding the fragment of human amyloid precursor protein,Aβ_(42,) was amplified from a human brain cDNA library by PCR withprimer 5′-AAGATGGATG CAGAATTCCG ACATGACTCA GGA-3′ (SEQ ID NO: 1) and5′TTAATGATGA TGATGATGAT GCGCTATGAC AACACCGCCCAC CATGAGTC CAAT-3′ (SEQ IDNO: 2). cDNAs of Aβ₄₂ and Dp35 were separately sequenced and subclonedinto the pUAST vector which was used to microinject to generatetransgenic flies. Embryos of the A23/+ genotype fly were microinjectedand 10 transgenic lines were obtained by standard methods (Rubin andSpradling, 1982, Science 218:348). The transgenic lines which showedobvious red-eye phenotypes were selected. Standard genetic balancers andchromosomes were used as described (Lindsley and Zimm, 1992, The genomeof Drosophila melanogaster (San Diego: Academic Press). Abl-1 andDf(3L)stj7, Ki strand flies were provided from Bloomington DrosophilaStock Center (Bloomington, Ind.) and crossed with transgenic flies toobtain Abl-/- background flies with ectopically expressed proteins.Double transgenic flies which expressed both Aβ₄₂ and Dp35-HA were alsogenerated by sequential crossing. All ectopically expressed Aβ₄₂ andDp35-HA were expressed under the control of gmr-GAL4 in fly eyes.

Example 8 Aβ₄₂ Activates Abl Kinase and Induces Drosophila NeuronalDeath

To determine whether Abl kinase activity is modulated by Aβ₄₂ inneurons, Drosophila neuronal cells, BG2-c2 (Ui-Tei et al,1996, Neurosci.Lett. 203:191) were treated with Aβ₄₂ (2 μM) and Abl kinase activity wasindirectly evaluated by monitoring the tyrosine-phosphorylation level ofEna, a kinase substrate that was previously found to be primarilyphosphorylated by Abl in Drosophila cells (Gertler et al., 1995, GenesDev. 9:521; Juang and Hoffmann, 1999, Oncogene 18:5138). Notably, Enaphosphorylation levels were elevated by Aβ₄₂ but reduced by STI571(Gleevec, Imatinib) (20 μM) (FIG. 1A). The result shows Abl kinaseactivity was stimulated by Aβ₄₂.

To determine whether activated Abl kinase was necessary for Aβ₄₂ inducedneuronal death, Abl kinase activity was suppressed by STI571 in Aβ₄₂treated neurons and cell survival was monitored using the assaydescribed above. FIG. 1B shows that the treatment with STI571 rescuedAβ₄₂-induced neuronal death. Consistently, reduction of Abl proteinexpression by dsRNA also prevented Aβ₄₂-induced neuronal death (FIG.1C). The dsRNA-abl markedly suppressed the Abl expression in adosage-dependent manner and was effective in cells treated with Aβ₄₂(FIG. 1D). No detectable viability change was observed after treatmentwith control dsRNA-EGFP. These data suggest that Abl kinase is involvedin the Aβ₄₂-triggered Drosophila neuronal death.

Example 9 Activated Abl Kinase Allows for Abl-Cdk5 Interaction and Cdk5Activation

To determine if Abl was an upstream regulator of cdk5 activationAβ₄₂-triggered Abl activation was investigated to determine if it couldaffect the Abl-cdk5 physical association. The endogenous Cdk5 wasco-immunoprecipitated with exogenous HA-tagged Abl in BG2-c2 cells todetermine the physical interaction between Abl and Cdk5. The complex ofAbl and Cdk5 was enhanced upon Aβ₄₂ treatment (lane 4 in FIGS. 2A and2B). Moreover, suppression of Abl kinase activity with STI571dramatically abolished the Abl-Cdk5 association in cells (lane 5 inFIGS. 2A and 2B). These results suggest that the Abl-Cdk5 interaction iscorrelated to the Aβ₄₂-initiated Abl kinase activity.

Whether Cdk5 was activated in the Abl-Cdk5 complex was alsoinvestigated. Cdk5 was immunoprecipitated from the cell lysate for anvitro kinase assay, using histone H1 as a substrate (Connell-Crowley etal., 2000, Curr. Biol. 10:599). Similar to results reported formammalian cells (Alvarez et al., 2001, Exp. Cell Res. 264:266),Drosophila Cdk5 kinase activity was elevated following Aβ₄₂ treatment ascompared to controls (FIG. 2B, bottom panel). The elevated Cdk5 kinaseactivity was consonant with the activated Abl kinase because suppressionof Abl kinase activity with STI571 not only dissociated the Abl-Cdk5interaction, but also sharply diminished Cdk5 kinase activity (FIG. 2B).Moreover, other Abl kinase regulating agents including doxorubicin (DX)and erbstatin analog (EA) were used to activate or suppress Abl kinaseactivity, respectively, and confirmed that Abl kinase activity wasrequired for modulation of Cdk5 kinase. Taken together, these resultsdemonstrate that Abl kinase activity is required for Abl-Cdk5interaction and Cdk5 activation in Aβ₄₂-triggered neurodegeneration.

Example 10 Genetic and Cell-Based Assays Show Abl is Essential inActivating Cdk5 Kinase for Aβ₄₂-Triggered Neurodegeneration

To demonstate that Abl is essential in activating Cdk5 kinase forAβ₄₂-triggered neurodegeneration in vivo, a transgenic fly model whichectopically expresses Aβ₄₂ in the developing compound eye wasestablished. By crossing the UAS-Aβ₄₂transgenic flies with theeye-specific gmr-GAL4 driver in either wild-type or Abl mutantbackgrounds (Df(3L)stj7, Ki/abl[1]) (Bennett and Hoffmann, 1992,Development 116:953), the role of Abl in neurodegeneration based on theseverity of eye phenotypes in larval and adult stages was tested.Neurodegenerative phenotypes were determined using TUNEL staining toreveal the neuronal apoptosis (see, e.g., McPhie et al., 2003, J.Neurosci. 23:6914). In the wild-type background, expression of Aβ₄₂behind the morphogenetic furrow in the 3rd-instar larval eye discsresulted in severe apoptosis in posterior region of the eye disccompared to gmr-GAL4 control (compare FIGS. 3A-b and 3A-a). Notably, anAbl mutant led to suppression of Aβ₄₂-induced apoptosis (FIG. 3A). TheAβ₄₂-induced apoptotic cells in the larval eye discs subsequently led tofacet disorder (fused ommatidium) and bristle lost in adult compoundeyes (FIG. 3B). Similarly, the Aβ₄₂-induced adult compound eyephenotypes were suppressed by mutation of Abl (FIG. 3B). These geneticresults suggest that Abl is an important factor contributing to Aβ₄₂induced Drosophila neurodegeneration.

To understand the role of Drosophila Cdk5 in neurodegeneration, thephosphorylation of Cdk5-Y15 was investigated to determine if it wascorrelated with the pathological activation of Cdk5 in neuronal cells.Cdk5-Y15 phosphorylation (Cdk5-pY15) levels in BG2-c2 cells wereexamined over time following treatment with 2 μM Aβ₄₂. The presence ofCdk5-pYll15 in the immunoprecipitates was shown by immunoblotting withan anti-phospho antibody specific for Cdk5-pY15. Within 60 minutes, Aβ₄₂caused an elevation of Cdk5-pY15, which was coincident with an increasein Cdk5 kinase activity (FIG. 4A). A kinase-inactive Cdk5 (Cdk5-Y15F) astransfected into the Aβ₄₂-stimulated neuronal cells and demonstratedthat the Cdk5-pY15 signal and neuronal death were diminished. Theseresults show that Abl is a Cdk5-Y15 kinase which triggers Cdk5 kinaseactivity in Aβ₄₂-stimulated neurons. To confirm this result, Abl kinaseactivity was suppressed with STI571 in Aβ₄₂-treated BG2-c2 cells. Theresult showed that both the Cdk5-pY15 and kinase activity werediminished (FIG. 4B). Moreover, dsRNA-abl (20 μg/2×10⁶ cells) wastransfected into BG2-c2 cells to reduce Abl expression two days beforeAβ₄₂ treatment. Consistent with the results obtained with STI571, thesecells exhibited a reduction of Cdk5-pY15 and kinase activity (FIG. 4C),confirming Abl is crucial for Aβ₄₂-induced Cdk5 activation.

This finding was further substantiated in vivo by immunoprecipitatingendogenous Cdk5 from wild type and Abl mutant adult fly heads to comparetheir Cdk5-pY15 levels and Cdk5 kinase activities. The Cdk5-pY15 signaland kinase activity were both elevated in flies overexpressing Aβ₄₂, ascompared to control flies (FIG. 4D, lanes 1 and 2). In contrast,overexpressing Aβ₄₂ in abl mutant flies resulted in the reduction ofCdk5-pY15 signal and Cdk5 kinase activity (FIG. 4D, lanes 3 and 4). Insummary, Abl is involved in Aβ₄₂-induced neurodegeneration via theregulation of Cdk5.

Example 11 p35 is not Cleaved into p25 in the DrosophilaNeurodegeneration Model

Like Abl, p35 is conserved in Drosophila and functions to modulate Cdk5activity during neurite outgrowth (Connell-Crowley et al., 2000, Curr.Biol. 10:599). In the AD brain, mammalian p35 is cleaved into p25 bycalpain to deregulate Cdk5 (Lee et al., 2000, Nature 405:360). Althougha putative calpain is also present in Drosophila (Jekely and Friedrich,1999, J. Biol. Chem. 274:23893), the consensus calpain cleavage site isabsent in Drosophila p35 (Dp35) (FIG. 5A). Therefore, it was unclearwhether Dp35 would be converted into p25 upon Aβ₄₂ stimulation. Toinvestigate this possibility, carboxyl-terminal HA-tagged Dp35 wastransfected into BG2-c2 cells before treating them with Aβ₄₂orA23187/Ca²⁺ (calcium ionophore). Intriguingly, no noticeable cleavage orreduction of Dp35 protein levels in the lysates were observed (FIG. 5B,left panel). To analyze the Dp35 cleavage in vivo, transgenic fliesco-expressing Dp35 and Aβ₄₂ in the developing eyes were generated usinga gmr-GAL4 driver. Consistent with the in vitro result, no visiblecleavage or reduction of the Dp35 protein in the Aβ₄₂ transgenic flieswere observed (FIG. 5B, right panel). These results show that Dp25 orother truncated forms of Dp35 protein is not required for theAβ₄₂-induced Drosophila neurodegeneration. However, the absence of Dp35cleavage in neurodegenerative flies does not necessarily imply that p35is dispensable for the regulation of Cdk5 in Drosophilaneurodegeneration.

To explore whether Dp35 is necessary for Aβ₄₂-induced Cdk5 activation inDrosophila, dsRNA-Dp35 was transfected into neuronal cells prior tostimulating cells with Aβ₄₂. The Dp35 protein expression levels weremarkedly suppressed by dsRNA-Dp35 in a dose-dependent manner, while nooff-target, non-specific effect was observed (FIG. 6A, left panel).Although Cdk5-Y15 was still phosphorylated in response to Aβ₄₂, the Cdk5kinase activity was virtually abolished after Dp35 was depleted fromcells (FIG. 6A, right panel). This result demonstrates that, while Dp35is not essential for Abl phosphorylation of Cdk5-Y15, the induction ofCdk5 by Abl still requires the involvement of p35.

To address the cooperative role of Abl and p35 in neurodegeneration, agenetic interaction assay was conducted. Overexpression of Dp35 in thedeveloping photoreceptor cells by a gmr-GAL4 driver resulted in apparentapoptosis in the 3rd-instar larval eye discs (FIG. 6B-b) and severeadult rough eye phenotypes (FIG. 6C-b). Consistent with the results fromAβ₄₂-induced eye phenotypes, the Dp35-induced neuronal apoptosis in theeye disc and compound eyes were suppressed by the Abl mutant (FIGS. 6Band 6C). Because overexpression of Dp35 was shown to induce Cdk5hyperactivation (Connell-Crowley et al, 2000, Curr. Biol. 10:599), thefinding that the Abl mutant suppressed Dp35-induced neurodegenerationfurther substantiates the role Abl plays in conjunction with Cdk5 inneurodegeneration.

Example 12 Cdk5-Y15 Phosphorylation by Abl is Required for Aβ₄₂-InducedCdk5 Subcellular Translocation

It was postulated that the amino-terminal myristoylation sequence ofmammalian p35 is the key domain for anchoring the Cdk5-p35 complex tothe cell membrane. Removal of the p35 amino-terminus, producing p25,would sever the complex's association with the cell membrane and thustranslocalize Cdk5 to cytosol (Patrick et al., 1999, J. Biol. Chem.273:24057). Intriguingly, although the cleavage of p35 into p25 may beabsent in drosophila neurodegenerative neurons, the myristoylation siteis conserved in Dp35 (FIG. 5A). The unique sequence feature of Dp35implies that Drosophila Cdk5-p35 complex may be resident on the cellmembrane and cannot be shuttled to the cytosol. To explore this,endogenous Cdk5 protein localization was examined by immunostaining.Without the stimulus of Aβ₄₂, the Cdk5 was primarily expressed on thecell membrane with lesser amounts found in the cytosolic region (FIG.7A, panel a). This finding is similar to the result observed inmammalian cells (Matsushita et al., 1995, Neuroreport 6:1267).Unexpectedly, one hour after a 2 μM Aβ₄₂stimulus, Cdk5 was markedlytranslocalized from cell membrane to the perinuclear cytoplasm (FIG. 7A,panel b), which suggests a p25-independent mechanism for Cdk5translocalization. By treating cells with Aβ₄₂ simultaneously with BL-1(Cdk5 kinase inhibitor) to block the Cdk5 activation, thetranslocalization of Cdk5 to the perinuclear cytoplasm was abrogated(FIG. 7A, panel c). This result indicated a kinase-dependent regulationof Cdk5 translocalization. Moreover, we discovered that the Cdk5 proteinwhich had been translocalized to the perinuclear cytoplasm could beshuttled back to plasma membrane by suppressing Cdk5 kinase activity onehour after Aβ₄₂ treatment (FIG. 7A, panel d). These results suggest thatDrosophila Cdk5, in response to Aβ₄₂ stimulus, can be reversiblyshuttled between the cell membrane and perinuclear cytoplasm by ap25-independent mechanism.

Since phosphorylation of Cdk5-Y15 correlates with Cdk5 activity, whetherthe Cdk5-pY15 is required for directing Cdk5 subcellular localizationwas investigated. Enhanced green fluorescent protein (EGFP) wasco-transfected with the Cdk5-Y15F mutation plasmid to label the neuronswith exogenous Cdk5-Y15F protein expression. Without the Aβ₄₂ stimulus,Cdk5-Y15F was primarily localized to the cell membrane similar to theresult seen with the wild-type Cdk5 (FIG. 7B, panel e). However,treatment of Aβ₄₂ did not noticeably affect the subcellular localizationof Cdk5-Y15F (FIG. 7B, panel f, arrow), in contrast to the untransfectedcells (FIG. 7B, panel f, arrowhead). Thus, the translocation of Cdk5 inresponse to A(342 stimulus may be modulated by the Abl phosphorylationof Cdk5-Y15. To test this idea, Abl kinase activity was suppressed withSTI571 before stimulating cells with Aβ₄₂ and immunostained both theAbl-HA and the endogenous Cdk5 proteins in the cells. Abl and Cdk5 werecolocalized to the plasma membrane and cytoplasm in control cells (FIG.7C, panels i, l, and o). Upon stimulation with Aβ₄₂, Cdk5 colocalizedwith Abl to the perinuclear cytoplasm (FIG. 7C, panels j, m, and p).However, such protein translocalization was markedly suppressed when Ablactivity was blocked (FIG. 7C, panels k, n, and q, arrow). dsRNA-abl wastransfected into neuronal cells and found a result similar to that seenwith STI571. These results accord well with the role of Abl inducedmodulation of Cdk5 and indicate a strong link between Abl regulation ofCdk5 and neurodegeneration.

Example 13 Aβ₄₂-Triggered Human p35 Cleavage into p25 is Insufficient toInitiate Cdk5 Kinase Activity in Cells Deficient of Abl Kinase Activity

To determine if the role of Abl in Drosophila neurodegeneration isconserved in mammals, Abl kinase activity in Aβ₄₂-induced mammalianneuronal apoptosis was investigated. Abl kinase was suppressed withSTI571 in human IMR-32 cells 24 hours prior to treatment with Aβ₄₂, andneuronal viability was determined. In accord with the result inDrosophila model system, the inhibition of Abl kinase activity rescuedthe neuronal death in IMR-32 cells (FIG. 8A). Formation of p25 has beenconsidered a causative agent that contributes to the deregulation ofCdk5 in human AD (Lee et al., 2000, Nature 405:360; Patrick et al.,1999, Nature 402:615). However, results of the Drosophilaneurodegeneration model suggest that Abl may be critical in regulatingCdk5. Thus, it was important to determine whether the conversion of p35into p25 is sufficient to deregulate Cdk5 activity while Abl activationis inhibited. After pretreating with STI571 for 24 hours to block Ablkinase activity, IMR-32 cells were incubated with Aβ₄₂to induce thecleavage of human p35. The cells were then assayed for Cdk5 kinaseactivity and Y-15 phosphorylation. The results obtained were in accordwith previously reported findings and showed that Aβ₄₂triggered thecleavage of p35 into p25, increased Cdk5-pY15 and increased kinaseactivity (FIG. 8B, lane 2) (Alvarez et al., 2001, Exp. Cell Res.264:266). However, in the presence of STI571, both the Cdk5-pY15 andkinase activation were abolished, though the formation of p25 was notobviously affected (FIG. 8B, lane 3). This result indicates that thecleavage of p35 into p25 is insufficient to activate Cdk5 while Ablkinase activity is suppressed.

To determine whether p25 functions cooperatively with Abl to assist themaximal activation of Cdk5 kinase (because the p25-Cdk5 complex is morestable than that of p35-Cdk5 (Patrick et al, 1998, J. Biol. Chem.273:24057; Tarricone et al, 2001, Mol. Cell 8:657)), a calpain inhibitor(Calpeptin) (Calbiochem San Diego, Calif.) was used to inhibit thecleavage of p35 in Aβ₄₂-treated IMR-32 cells. Interestingly, theblockade of p35 cleavage did not diminish Cdk5-pY15 but the Cdk5activity was considerably reduced (FIG. 8C, compare lanes 2 and 3),indicating that the formation of p25 is indeed important for the Cdk5activation whereas the phosphorylation of Cdk5-Y15 is independent of p35cleavage. In addition, we also found that the co-treatment of cells withCalpeptin and STI571 further reduced the Cdk5 kinase activity (FIG. 8C,compare lane 5), confirming that Abl and p25 function cooperatively inderegulating Cdk5 in mammalian cells. Taken together, this datademonstrates that Abl is a critical component, which mediates Cdk5-pY15and Cdk5 activation in Aβ₄₂-triggered neurodegeneration, and thecleavage of p35 into p25 functions to stabilize the Abl-Cdk5 complex forthe maximal induction of Cdk5 activity.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupercede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

1. A method of treating a subject having a neurodegenerative diseasecomprising administering to the subject, an agent which inhibits Ablkinase activity.
 2. The method of claim 1, wherein the neurodegenerativedisease is Alheimer's disease.
 3. The method of claim 1, wherein theagent is a polypeptide, a nucleic acid, or a small molecule.
 4. Themethod of claim 3, wherein the nucleic acid is an RNA molecule.
 5. Themethod of claim 4, wherein the RNA molecule is siRNA molecule.
 6. Themethod of claim 4, wherein the RNA molecule is ds-RNA Abl.
 7. The methodof claim 3, wherein the small molecule is STI571.
 8. The method of claim3, wherein the agent is an erbstatin analog.
 9. The method of claim 1,wherein the subject is a human.
 10. The method of claim 1, wherein theAbl kinase activity inhibited is phosphorylation of a substrate.
 11. Themethod of claim 10, wherein the substrate is another kinase.
 12. Themethod of claim 11, wherein the substrate is a cyclin dependent kinase.13. The method of claim 12, wherein the cyclin dependent kinase iscyclin dependent kinase 5 (Cdk5).
 14. A method of inhibitingdegeneration of a neuron comprising contacting the neuron with an agentwhich inhibits Abl kinase activity.
 15. The method of claim 14, whereinthe neuron is a mammalian neuron.
 16. The method of claim 15, whereinthe mammalian neuron is a human neuron.
 17. The method of claim 14,wherein the neuron is a Drosophila melanogaster neuron.
 18. The methodof claim 14, wherein the agent is a polypeptide, a nucleic acid, or asmall molecule.
 19. The method of claim 18, wherein the nucleic acid isan RNA molecule.
 20. The method of claim 19, wherein the RNA molecule issiRNA molecule.
 21. The method of claim 19, wherein the RNA molecule isds-RNA Abl.
 22. The method of claim 14, wherein the small molecule isSTI571.
 23. The method of claim 14, wherein the agent is an erbstatinanalog.
 24. A method of inhibiting formation of a complex comprisingAbl, Cdk5, Cables, Abi and p35 or a fragment thereof, in a neuroncomprising, contacting the neuron with an agent which inhibits Ablkinase activity.
 25. The method of claim 24, wherein the neuron is amammalian neuron.
 26. The method of claim 25, wherein the mammalianneuron is a human neuron.
 27. The method of claim 24, wherein the neuronis a Drosophila melanogaster neuron.
 28. The method of claim 24, whereinthe agent is a polypeptide, a nucleic acid, or a small molecule.
 29. Themethod of claim 28, wherein the nucleic acid is an RNA molecule.
 30. Themethod of claim 29, wherein the RNA molecule is siRNA molecule.
 31. Themethod of claim 29, wherein the RNA molecule is ds-RNA Abl.
 32. Themethod of claim 28, wherein the small molecule is STI571.
 33. The methodof claim 28, wherein the agent is an erbstatin analog.
 34. The method ofclaim 24, wherein the Abl kinase activity inhibited is phosphorylationof a substrate.
 35. The method of claim 34, wherein the substrate isanother kinase.
 36. The method of claim 35, wherein the substrate is acyclin dependent kinase.
 37. The method of claim 36, wherein the cyclindependent kinase is cyclin dependent kinase
 5. 38. A method ofinhibiting Cdk5 activity in a cell comprising contacting the cell withan agent which inhibits Abl kinase activity thereby inhibiting Cdk5activity.
 39. The method of claim 38, wherein the agent is apolypeptide, a nucleic acid, or a small molecule.
 40. The method ofclaim 39, wherein the nucleic acid is an RNA molecule.
 41. The method ofclaim 40, wherein the RNA molecule is siRNA molecule.
 42. The method ofclaim 41, wherein the RNA molecule is ds-RNA Abl.
 43. The method ofclaim 39, wherein the small molecule is STI571.
 44. The method of claim39, wherein the agent is an erbstatin analog.
 45. The method of claim38, wherein the Abl kinase activity inhibited is phosphorylation of asubstrate.
 46. A method of screening for an agent which treats aneurodegenerative disease comprising a) contacting a cell with theagent; and b) detecting at least one of Abl kinase activity in the celland changes in Cdk5 subcellular localization where a decrease in Ablkinase activity in the cell or a change in Cdk5 subcellular localizationcontacted with the agent compared to a cell not contacted with the agentindicates that the agent can be used to treat a neurodegenerativedisease.
 47. The method of claim 46, wherein the neurodegenerativedisease is Alheimer's disease.
 48. The method of claim 46, wherein theagent is a polypeptide, a nucleic acid, or a small molecule.
 49. Themethod of claim 46, wherein the Abl kinase activity is phosphorylationof a substrate.
 50. The method of claim 46, wherein the cell is aneuron.
 51. The method of claim 50, wherein the neuron is a mammalianneuron.
 52. The method of claim 51, wherein the mammalian neuron is ahuman neuron.
 53. The method of claim 50, wherein the neuron is aninsect neuron.
 54. The method of claim 53, wherein the insect neuron isa drosophila melanogaster neuron.
 55. A method of screening for an agentwhich inhibits degeneration of a neuron comprising contacting a cellwith the agent and detecting at least one of the Abl kinase activity inthe cell and Cdk5 protein subcellular localization, where a decrease inAbl kinase activity in the cell contacted with the agent or a change inCdk5 protein subcellular localization compared to a cell not contactedwith the agent indicates that the agent can be used to inhibitneurodegeneration.
 56. The method of claim 55, wherein the agent is apolypeptide, a nucleic acid, or a small molecule.
 57. The method ofclaim 55, wherein the Abl kinase activity is phosphorylation of asubstrate.
 58. The method of claim 55, wherein the cell is a neuron. 59.The method of claim 58, wherein the neuron is a mammalian neuron. 60.The method of claim 59, wherein the mammalian neuron is a human neuron.61. The method of claim 60, wherein the neuron is an insect neuron. 62.The method of claim 61, wherein the insect neuron is a drosophilamelanogaster neuron.
 63. A transgenic animal comprising a transgenecomprising a nucleotide sequence encoding a β amyloid protein.
 64. Thetransgenic animal of claim 63, wherein the animal is drosophilamelanogaster.
 65. The transgenic animal of claim 63, wherein the βamyloid protein is Aβ₄₂.
 66. The transgenic animal of claim 65, whereinthe β amyloid protein is ectopically expressed.
 67. The transgenicanimal of claim 66, wherein the β amyloid protein is ectopicallyexpressed in the compound eye.
 68. The transgenic animal of claim 63,wherein the transgenic animal expresses a mutant form of Abl.
 69. Thetransgenic animal of claim 68, wherein the transgenic animal expresses awild type form of Abl.
 70. The transgenic animal of claim 69, whereinthe transgenic animal comprises a nucleotide sequence encoding GMR-gal4.